The long-range objective of our studies is to determine the role of the CRES (cystatin-related epididymal spermatogenic) protein in reproductive function. Gene and protein structure studies show that the CRES protein is a new member of the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, several lines of evidence suggest that CRES may not function as a classic cystatin. Firstly, CRES protein lacks two of three consensus sites important for inhibition of cysteine proteases. Secondly, our in vitro enzyme assays show that, in contrast to cystatin C, CRES protein does not inhibit the cysteine proteases papain, cathepsin B, legumain, or caspase. Finally, unlike the ubiquitous expression of the family 2 cystatins, CRES protein expression is unique and is restricted to sites of high proteolytic activity within specific reproductive tissues including the proximal caput epididymis, anterior pituitary gonadotropes, and sperm acrosomes. Besides being localized to sites of high proteolytic activity, very strong evidence that CRES protein is a protease inhibitor is our recent in vitro enzyme assays showing that CRES protein is inhibitory against members of a family of highly substrate specific serine proteases known as the prohormone convertases. This recently discovered family of proteins play critical roles in prohormone and proprotein processing in a variety of organ systems including the neuroendocrine and reproductive systems. Taken together, our preliminary studies provide strong evidence that CRES protein is a potential inhibitor of an important family of proteases with widespread biological significance and therefore further studies on CRES protein are warranted. Because the epididymis and sperm acrosome are active sites where precursor proteins are processed to their mature forms and in vitro, CRES inhibits proteases with confirmed biological roles in protein processing, we hypothesize that CRES protein is a novel prohormone convertase inhibitor which regulates proteolytic processing events in the sperm acrosome and epididymis and thus is important for fertilization and sperm maturation. We will test our hypothesis in the following specific aims: 1) examine CRES protein as a convertase inhibitor by in vitro enzyme assays and co- immunoprecipitation experiments. Functional analyses of CRES as a convertase inhibitor will be initiated by examining potential targets of convertases; 2) examine acrosomal CRES protein in mouse sperm function; and 3) examine the biological function of CRES protein in vivo by gene knock-out studies.